Lymphocyte subpopulations (e.g., T cells and B cells) can be distinguished by their biological functions and also by characteristic cell surface proteins which are involved in lymphocyte-specific signal transduction and/or pathogenesis of lymphotropic viruses. Some of these lymphocyte transduction proteins have been identified with specific antibodies. These cell surface antigenic markers are commonly referred to as "cluster of differentiation" or "CD" markers. Moreover, the expression of some of these surface markers correlates with the stage of differentiation of particular lymphocyte subpopulations. Various CD markers have been identified and classified in human and nonhuman lymphocyte populations (see, Immunology: A Synthesis, Second Ed., Golub, E. S. and Green, D. R., Eds., Sinauer Associates, Sunderland, Mass., 1991, Appendix 2, incorporated herein by reference). When, for example, a murine marker has been found to be homologous to a particular human marker, both markers receive the same CD designation. Although lymphocyte transduction proteins from various species may share some degree of sequence homology, it is not known whether homologous lymphocyte transduction molecules perform equivalent roles between species or whether homologous lymphocyte transduction molecules are functionally interchangeable.
In addition to correlating with lymphocyte subpopulations, certain CD molecules also have been found to play important roles in transduction of signals for lymphocyte activation and/or in transduction of viral infectivity (e.g., virion attachment and entry) and/or viral pathogenesis. For example, CD2, CD4, and CD8 each play a role in T cell activation, and CD4 also is involved in HIV pathogenesis and may function as a virus receptor for HIV-1. For example, CD4 is expressed on helper T cells and plays an important signalling role during antigen recognition by binding to a nonpolymorphic region of major histocompatibility complex (MHC) class II molecules (Glaichenhaus et al. (1991) Cell 64: 511; Littman, D. R. (1987) Ann. Rev. Immunol. 5: 561; Parnes, J. R. (1989) Adv. Immunol. 44: 265; Barzaga-Gilbert et al. (1992) J. Exp. Med. 175: 1707). It is believed that CD4, in conjunction with MHC class II molecules, plays an essential role in the selection of the T cell repertoire during thymic ontogeny (Teh et al. (1991) Nature 349: 241; Robey et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 608). Evidence suggests that CD4 is important in MHC class II recognition, and during antigen recognition CD4 interacts with the T cell antigen receptor-CD3 complex and the lymphocyte-specific tyrosine kinase p56lck (Sancho et al. (1992) J. Biol. Chem. 267: 7871; Pelchen-Matthews et al. (1992) J. Cell. Biol. 117: 279; Collins et al. (1992) J. Immunol. 148: 2159; Haughn et al. (1992) Nature 358: 328). The functional association between CD4, p56lck, and other lymphocyte surface molecules such as MHC class II molecules indicates a potential role for CD4.sup.+ T cells in mediating autoimmune diseases as well as other immune disorders. The interaction between CD4 and membrane-associated p56lck has also been implicated in HIV-1 pathogenesis (Crise B. and Rose J. K. (1992) J. Virol. 66: 2296).
Several CD antigens and other lymphocyte surface molecules are involved in lymphocyte transduction, which comprises activation signal transduction and/or viral transduction in lymphocytes. Lymphocyte activation signal transduction refers to the signal transduction pathway(s) which produce antigenic activation of lymphocytes, from antigen recognition to proliferation of antigen-specific cells and/or acquisition of specific biological functions (e.g., IL-2 expression). For example, the interaction of antigen with the antigen receptor on T lymphocytes initiates an ordered series of pleiotropic changes; a process denoted as T lymphocyte activation. T lymphocyte activation is a 7 to 10 day process that results in cell division and the acquisition of immunological functions such as cytotoxicity and the production of lymphokines that induce antibody production by B lymphocytes and control the growth and differentiation of granulocyte and macrophage precursors. The cytokines produced by activated T lymphocytes act upon other cells of the immune system to coordinate their behavior and bring about an effective immune response.
The initiation of T lymphocyte activation requires a complex interaction of the antigen receptor with the combination of antigen and self-histocompatibility molecules on the surface of antigen-presenting cells. T lymphocytes may also be activated by relatively simple stimuli such as the combination of a calcium ionophore (e.g., ionomycin) and an activator of protein kinase C, such as phorbol myristate acetate (PMA). Several lectins, including phytohemagglutinin (PHA) may also be used to activate T cells (Nowell, Cancer Res. 20:462-466 (1960)). T lymphocyte activation involves the specific regulation of particular subsets of genes. The transcriptional regulation characteristic of T cell activation begins minutes after the antigen encounter and continues until at least 10 days later. The T lymphocyte activation genes can be grouped according to the time after stimulation at which each gene is transcribed. Early genes are the first subset of T lymphocyte activation genes that is expressed during the activation process. Expression of the early genes triggers the transcriptional modulation of subsequent genes in the activation pathway.
CD8 is expressed on the surface of cytotoxic T cells and function in signal transduction of antigen recognition by MHC class I-specific T cell receptors (TCRs). CD2 is expressed on the surface of T cells and mediates adhesion of T cells to antigen-presenting cells (APCs) by interacting with the APC surface molecule LFA-3, facilitating antigen recognition by T cells. CD3 forms a complex with the T cell receptor which binds specific antigen as a primary step in antigenic activation of T cells.
Helper and cytotoxic subsets of T lymphocytes can be distinguished by their surface expression of the CD4 and/or CD8 glycoproteins. Immature thymocytes show a coordinate expression of both CD4 and CD8, but mature lymphocytes express either CD4 or CD8, but not both. Thus, the repression of either CD4 or CD8 may be a pivotal event in the differentiation of T cells into functionally distinct subsets. CD4 expression is associated with T helper lymphocytes, whereas CD8 expression is associated with T cytotoxic lymphocytes. Both CD4 and CD8 are considered to be members of the immunoglobulin gene superfamily on the basis of sequence homology, but not on the basis of biological function. CD4 is a 55 kD glycoprotein having a extracellular domain that is 372 amino acids long composed of four tandem Ig-like VJ regions, a 23 amino acid long transmembrane domain, and a 38 amino acid long cytoplasmic domain. CD8 generally exists as a heterodimer of two disulfide-linked subunits, .alpha. (34-38 kD) and .beta. (30-35 kD), or as .alpha.--.alpha. homodimers.
Besides antigenic activation of T cells, lymphocyte transduction may alternatively or additionally comprise viral transduction in lymphocytes, wherein a lymphocyte transduction protein is necessary for efficient expression of a viral-induced phenotype in a lymphocyte or lymphocyte population. For example, CD4 is involved in the pathogenesis of HIV-1 in producing acquired immune deficiency syndrome (AIDS) and other HIV-induced immune system disorders. Although CD4 has been proposed as a receptor for attachment and infectious entry of HIV virions into CD4.sup.+ T cells, CD4 may also function in transducing HIV-induced signals to a CD4.sup.+ T cell by a mechanism that does not require infection of the T cell by HIV-1 (Groux et al. (1992) J. Exp. Med. 175: 331).
Some other CD antigens also have been shown to be targets for adsorption and entry of lymphotropic viruses. For example, CD21 mediates infection of human B lymphocytes with the Epstein-Barr virus (EBV) (Hedrick et al. (1992) Eur. J. Immunol. 22: 1123). The gp120 envelope glycoprotein of HIV binds to the human CD4 molecule on the surface of T lymphocytes and some macrophage-monocytes and appears to mediate internalization of the HIV virion (Arthos, J. et al. (1989) Cell 57: 469). Indeed, HIV infection is characterized by a dramatic decline in the number of CD4.sup.+ T cells, which may result in some of the pathological changes noted in AIDS (e.g., opportunistic infections, increased incidence of neoplasms). Neither HIV nor EBV infect mouse lymphocytes and thus there are presently no convenient nonhuman models for studying these viral infections or resultant immune system effects, such as the immunodeficiency of HIV infection which is characterized by deletion of CD4.sup.+ lymphocytes. It may not be necessary for HIV to infect T cells in order to produce pathogenesis; HIV virions and/or HIV-infected T cells may produce pathological effects on uninfected T cells (particularly helper T cells) by a non-infective mechanism (e.g., depletion of helper T cells may occur without the cells being infected with HIV). However, it appears that HIV-related pathological effects can be produced in T cells bearing human CD4 molecules, but not in nonhuman T cells which lack human CD4 (Groux et al. (1992) op.cit.). Thus, nonhuman models expressing human lymphocyte transduction proteins would be highly desirable for studying human immunodeficiencies and human viral pathogenesis.
CD44 participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis (Jackson et al. (1992) J. Biol. Chem. 267: 4732). CD25 is the .beta. chain of the IL-2 receptor. Several other T cell-specific membrane-associated proteins that are involved in lymphocyte activation and/or lymphotropic viral pathogenesis have also been identified and cloned (Weissman et al. (1988) Science 239: 1028; Hedrick et al (1984) Nature 308: 149; Chien et al (1984) Nature 312: 31).
In addition to several of the recognized CD antigens which have been defined by specific antibodies, other proteins function in lymphocyte activation and/or viral pathogenesis. For example, the lymphocyte-specific p56lck tyrosine kinase interacts with CD4 and participates in T cell activation and HIV-1 viral pathogenesis (Crise B. and Rose J. K. op.cit.; Collins op.cit.; Cefai et al. (1992) J. Immunol. 149: 285; Caron et al. (1992) Mol. Cell. Biol. 12: 2720). Molina et al. (1992) Nature 357: 161, report that thymocyte development is inhibited by an absence of functional p56lck protein. Other membrane-associated tyrosine kinases (e.g., p53/56lyn, p59fyn) may also participate in lymphocyte signal transduction (Campbell M. A. and Sefton B. M. (1992) Mol. Cell. Biol. 12: 2315).
Gene targeting, mediated by homologous recombination between a targeting polynucleotide construct and a homologous chromosomal sequence, has been used to disrupt several genes, including the HPRT gene, .beta.2-microglobulin gene, int-2 proto-oncogene, and the fos proto-oncogene (Thomas and Cappechi (1987) Cell 51: 503; Zijlstra et al. (1989) Nature 342: 435; Mansour et al. (1988) Nature 336: 348; and Johnson et al. (1989) Science 245: 1234: Adair et al. (1989) Proc. Natl. Acad. Sci (U.S.A.) 86:4574; Capecchi, M. (1989) TIG 5:70; Capecchi, M. (1989) Science 244:1288). Mansour et al. (1988) op.cit. have described homologous targeting constructs that include a HSV tk gene that permits negative selection against nonhomologous integration events in conjunction with positive selection for integrated transgenes.
Transgenic nonhuman mammalian cells and transgenic nonhuman animals which harbor one or more inactivated genes required for production of functional lymphocyte transduction molecules, such as CD4, are desirable as experimental model systems and as hosts for expression of transgenes encoding heterologous (e.g., human) cell surface proteins. Kucherlapati (WO91/10741) discusses strategies for producing non-human mammalian hosts characterized by inactivated endogenous immunoglobulin loci. Lonberg (WO92/03918) describes construction of vectors for targeting endogenous immunoglobulin loci and inactivation of endogenous immunoglobulin genes with such targeting vectors. Rahemtulla et al. (1991) Nature 353: 180, describes disruption of an endogenous murine CD4 gene by homologous gene targeting in embryonic stem cells. Jasin et al. (1990) Genes Devel. 4: 157, report targeting the human CD4 gene in a T lymphoma cell line by epitope addition. Koh et al. (1992) Science 256: 1210, report disruption of an endogenous murine CD8 gene by homologous gene targeting in ES cells. Molina et al. (1992) op.cit., describes disruption of the murine lck gene, which encodes a tyrosine kinase implicated in signal transduction by CD4 and CD8. Grusby et al. (1991) Science 253: 1417, describes disruption of the MHC Class II A.sup.b beta gene by gene targeting in mice; the resultant targeted mice are reported to be depleted of CD4.sup.+ lymphocytes.
Animals having a functionally disrupted endogenous lymphocyte transduction gene and also harboring a transgene which expresses a heterologous (i.e., derived from a different species) lymphocyte transduction gene product would be useful as models for studying disease pathogenesis and fundamental immunology, as well as providing useful models for screening for novel therapeutic agents to treat viral infection, autoimmune diseases, and immunosuppression. For example, nonhuman animals in which helper T cell development is dependent on expression of human CD4 would be useful for studies of several human diseases in which the function of CD4.sup.+ T cells is altered.
Based on the foregoing, it is clear that a need exists for nonhuman cells and nonhuman animals harboring one or more functionally disrupted endogenous lymphocyte transduction genes and also harboring a transgene encoding a cognate human lymphocyte transduction polypeptide which is expressed in at least a subset of host lymphocytes. Thus, it is an object of the invention herein to provide targeting transgenes for inactivating, by homologous recombination, endogenous lymphocyte transduction genes, particularly the CD4 gene. It is also an object of the invention to provide methods to produce transgenic nonhuman cells and transgenic nonhuman animals harboring correctly targeted homologously recombined transgenes of the invention.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.